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Proximity-dependent initiation of hybridization chain reaction.

Koos B, Cane G, Grannas K, Löf L, Arngården L, Heldin J, Clausson CM, Klaesson A, Hirvonen MK, de Oliveira FM, Talibov VO, Pham NT, Auer M, Danielson UH, Haybaeck J, Kamali-Moghaddam M, Söderberg O - Nat Commun (2015)

Bottom Line: This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product.In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry.As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical center, Husargatan 3, Box 815, SE-75108 Uppsala, Sweden.

ABSTRACT
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

No MeSH data available.


Flow-cytometric analysis of proxHCR on the model reaction of internalization of EGF/EGFR complex.Background levels of unstimulated cells (a) at 525 nm (AlexaFluor488) and 670 nm (Cy5). After 1 min of stimulation (b) with AlexaFluor488-EGF, a small shift at 525 nm can be observed. Furthermore, fluorescence at 670 nm shifts to higher values as well. After 10 min of stimulation (c), a large shift at 525 nm can be seen while fluorescence at 670 nm shifts back to background level. Prolonged incubation (30 min) did not alter the fluorescence levels significantly (d).
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f6: Flow-cytometric analysis of proxHCR on the model reaction of internalization of EGF/EGFR complex.Background levels of unstimulated cells (a) at 525 nm (AlexaFluor488) and 670 nm (Cy5). After 1 min of stimulation (b) with AlexaFluor488-EGF, a small shift at 525 nm can be observed. Furthermore, fluorescence at 670 nm shifts to higher values as well. After 10 min of stimulation (c), a large shift at 525 nm can be seen while fluorescence at 670 nm shifts back to background level. Prolonged incubation (30 min) did not alter the fluorescence levels significantly (d).

Mentions: To determine whether proxHCR would also work with a flow cytometry readout, we set up an assay to detect the binding of epidermal growth factor (EGF) to EGF receptor (EGFR) in A431 cells (Fig. 6). The proxHCR assay is used to detect membranous AlexaFluor488-EGF/EGFR at 670 nm (Cy5), whereas internalized complexes would be detected by the AlexaFluor488 attached to EGF. Analysis of the flow cytometry data shows a small shift of fluorescence at 525 nm (AlexaFluor488) after stimulation with AlexaFluor488-EGF for 1 min at 37 °C (Fig. 6a,b). This shift increases after 10 min at 37 °C (Fig. 6c). In contrast, we can see an increase of the Cy5 fluorescence after 1 min at 37 °C (Fig. 6a,b), which decreases to baseline after 10 min of stimulation (Fig. 6c). Neither fluorescence at 525 nm nor at 670 nm change considerably after 30 min of incubation with AlexaFluor488-EGF (Fig. 6d).


Proximity-dependent initiation of hybridization chain reaction.

Koos B, Cane G, Grannas K, Löf L, Arngården L, Heldin J, Clausson CM, Klaesson A, Hirvonen MK, de Oliveira FM, Talibov VO, Pham NT, Auer M, Danielson UH, Haybaeck J, Kamali-Moghaddam M, Söderberg O - Nat Commun (2015)

Flow-cytometric analysis of proxHCR on the model reaction of internalization of EGF/EGFR complex.Background levels of unstimulated cells (a) at 525 nm (AlexaFluor488) and 670 nm (Cy5). After 1 min of stimulation (b) with AlexaFluor488-EGF, a small shift at 525 nm can be observed. Furthermore, fluorescence at 670 nm shifts to higher values as well. After 10 min of stimulation (c), a large shift at 525 nm can be seen while fluorescence at 670 nm shifts back to background level. Prolonged incubation (30 min) did not alter the fluorescence levels significantly (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490387&req=5

f6: Flow-cytometric analysis of proxHCR on the model reaction of internalization of EGF/EGFR complex.Background levels of unstimulated cells (a) at 525 nm (AlexaFluor488) and 670 nm (Cy5). After 1 min of stimulation (b) with AlexaFluor488-EGF, a small shift at 525 nm can be observed. Furthermore, fluorescence at 670 nm shifts to higher values as well. After 10 min of stimulation (c), a large shift at 525 nm can be seen while fluorescence at 670 nm shifts back to background level. Prolonged incubation (30 min) did not alter the fluorescence levels significantly (d).
Mentions: To determine whether proxHCR would also work with a flow cytometry readout, we set up an assay to detect the binding of epidermal growth factor (EGF) to EGF receptor (EGFR) in A431 cells (Fig. 6). The proxHCR assay is used to detect membranous AlexaFluor488-EGF/EGFR at 670 nm (Cy5), whereas internalized complexes would be detected by the AlexaFluor488 attached to EGF. Analysis of the flow cytometry data shows a small shift of fluorescence at 525 nm (AlexaFluor488) after stimulation with AlexaFluor488-EGF for 1 min at 37 °C (Fig. 6a,b). This shift increases after 10 min at 37 °C (Fig. 6c). In contrast, we can see an increase of the Cy5 fluorescence after 1 min at 37 °C (Fig. 6a,b), which decreases to baseline after 10 min of stimulation (Fig. 6c). Neither fluorescence at 525 nm nor at 670 nm change considerably after 30 min of incubation with AlexaFluor488-EGF (Fig. 6d).

Bottom Line: This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product.In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry.As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical center, Husargatan 3, Box 815, SE-75108 Uppsala, Sweden.

ABSTRACT
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

No MeSH data available.